Ferritin ELISA. Enzyme immunoassay for the in-vitro-diagnostic quantitative determination of Ferritin in human serum.
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1 Instructions for Use Ferritin ELISA Enzyme immunoassay for the invitrodiagnostic quantitative determination of Ferritin in human serum. DB C I B L I N T E R N A T I O N A L G M B H Flughafenstrasse 52a Phone: +49 (0) IBL@IBLInternational.com D22335 Hamburg, Germany Fax: +49 (0)
2 INTENDED USE Enzyme immunoassay for the invitrodiagnostic quantitative determination of Ferritin in human serum. PRINCIPLE OF THE TEST The principle of the following enzyme immunoassay test follows a typical onestep capture or sandwich type assay. The assay makes use of two highly specific monoclonal antibodies: A monoclonal antibody specific for ferritin is immobilized onto the microplate and another monoclonal antibody specific for a different region of ferritin is conjugated to horse radish peroxidase (HRP). Ferritin from the sample and standards are allowed to bind simultaneously to the plate and to the HRP conjugate. The washing and decanting steps remove any unbound HRP conjugate. After the washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stopping solution. The absorbance is measured on a microtiter plate reader. The intensity of the colour formed by the enzymatic reaction is directly proportional to the concentration of ferritin in the sample. A set of standards is used to plot a standard curve from which the amount of ferritin in patient samples and controls can be directly read. CLINICAL APPLICATIONS Ferritin is a protein which serves as a storage center for iron. It is found in many tissues but the highest concentrations are in the liver, spleen and bone marrow. The total body iron stores in normal people correlate well with the concentration of ferritin in serum. If there is a deficiency in iron, that is the concentration of iron is low in the blood, the ferritin result will be decreased. Likewise, an overload of iron indicates an increase in the level of ferritin. However, in some conditions like liver disease ferritin will be elevated. PROCEDURAL CAUTIONS AND WARNINGS 1. Users should have a thorough understanding of this protocol for the successful use of this kit. Reliable performance will only be attained by strict and careful adherence to the instructions provided. 2. Control materials or serum pools should be included in every run at a high and low level for assessing the reliability of results. 3. When the use of water is specified for dilution or reconstitution, use deionized or distilled water. 4. In order to reduce exposure to potentially harmful substances, gloves should be worn when handling kit reagents and human specimens. 5. All kit reagents and specimens should be brought to room temperature and mixed gently but thoroughly before use. Avoid repeated freezing and thawing of reagents and specimens. 6. A calibrator curve must be established for every run. 7. The controls should be included in every run and fall within established confidence limits. 8. Improper procedural techniques, imprecise pipetting, incomplete washing as well as improper reagent storage may be indicated when assay values for the control do not reflect established ranges. 9. When reading the microplate, the presence of bubbles in the wells will affect the optical densities (ODs). Carefully remove any bubbles before performing the reading step. 10. The substrate solution (TMB) is sensitive to light and should remain colourless if properly stored. Instability or contamination may be indicated by the development of a blue colour, in which case it should not be used. 11. When dispensing the substrate and stopping solution, do not use pipettes in which these liquids will come into contact with any metal parts. 12. To prevent contamination of reagents, use a new disposable pipette tip for dispensing each reagent, sample, standard and control. 13. Do not mix various lot numbers of kit components within a test and do not use any component beyond the expiration date printed on the label. 14. Kit reagents must be regarded as hazardous waste and disposed of according to national regulations. V 10.0 May 15, /6
3 LIMITATIONS 1. All the reagents within the kit are calibrated for the direct determination of ferritin in human serum. The kit is not calibrated for the determination of ferritin in saliva, plasma or other specimens of human or animal origin. 2. Do not use grossly hemolyzed, grossly lipemic, icteric or improperly stored serum. 3. Any samples or control sera containing azide or thimerosal are not compatible with this kit, as they may lead to false results. 4. Only calibrator A may be used to dilute any high serum samples. The use of any other reagent may lead to false results. 5. The results obtained with this kit should never be used as the sole basis for clinical diagnosis. For example, the occurrence of heterophilic antibodies in patients regularly exposed to animals or animal products has the potential of causing interferences in immunological tests. Consequently, the clinical diagnosis should include all aspects of a patient s background including the frequency of exposure to animals/products if false results are suspected. 6. Some individuals may have antibodies to mouse protein that can possibly interfere in this assay. Therefore, the results from any patients who have received preparation of mouse antibodies for diagnosis or therapy should be interpreted with caution. SAFETY CAUTIONS AND WARNINGS POTENTIAL BIOHAZARDOUS MATERIAL Human serum that may be used in the preparation of the standards and control has been tested and found to be nonreactive for Hepatitis B surface antigen and has also been tested for the presence of antibodies to HCV and Human Immunodeficiency Virus (HIV) and found to be negative. No test method however, can offer complete assurance that HIV, HCV and Hepatitis B virus or any infectious agents are absent. The reagents should be considered a potential biohazard and handled with the same precautions as applied to any blood specimen. CHEMICAL HAZARDS Avoid contact with reagents containing TMB, hydrogen peroxide and sulfuric acid. If contacted with any of these reagents, wash with plenty of water. TMB is a suspected carcinogen. SPECIMEN COLLECTION AND STORAGE Approximately 0.2 ml of serum is required per duplicate determination. Collect 45 ml of blood into an appropriately labelled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at 4 C for up to 24 hours or at 10 C or lower if the analyses are to be done at a later date. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling. SPECIMEN PRETREATMENT This assay is a direct system; no specimen pretreatment is necessary. REAGENTS AND EQUIPMENT NEEDED BUT NOT PROVIDED 1. Precision pipettes to dispense 20, 50, 150, 200 and 300 μl 2. Disposable pipette tips 3. Distilled or deionized water 4. Orbital shaker (600 rpm) (e.g. EAS 2/4, SLT) or Linear shaker (200rpm) 5. Microplate reader with a filter set at 450 nm and an upper OD limit of 3.0 or greater* (see assay procedure step 10). V 10.0 May 15, /6
4 REAGENTS PROVIDED 1. MTP Microtiter Plate Mouse AntiFerritin Antibody Coated Break Apart Wells Ready To Use. Contents: One 96 well (12x8) monoclonal antibodycoated Microtiter Plate in a resealable pouch with desiccant. Storage: Refrigerate at 28 C 2. ENZCONJ CONC Enzyme Conjugate Concentrate (X50) Requires Preparation Contents: AntiFerritin monoclonal antibodyhrp conjugate in a proteinbased buffer with a nonmercury preservative. Volume: 600 μl/vial Storage: Refrigerate at 28 C Preparation: Dilute 1:50 in assay buffer before use (eg. 40 μl of HRP in 2 ml of assay buffer). If the whole plate is to be used dilute 480 μl of HRP in 24 ml of assay buffer. Discard any that is left over. 3. CAL A F Standards / Calibrators Ready To Use. Contents: Six vials containing ferritin in a proteinbased buffer with a nonmercury preservative. Prepared by spiking buffer with a defined quantity of ferritin. Calibrated against World Health Organization (WHO) 3rd IS 94/572. *Listed below are approximate concentrations, please refer to vial labels for exact concentrations. Calibrator Concentration Volume Calibrator A 0 ng/ml 2.0 ml Calibrator B 10 ng/ml 0.5 ml Calibrator C 50 ng/ml 0.5 ml Calibrator D 150 ng/ml 0.5 ml Calibrator E 400 ng/ml 0.5 ml Calibrator F 800 ng/ml 0.5 ml Storage: Stability: Refrigerate at 28 C 12 months in unopened vials or as indicated on label. Once opened, the standards should be used within 14 days or aliquoted and stored frozen. Avoid multiple freezing and thawing cycles. 4. CONTROL LOW & CONTROL HIGH Controls Ready To Use. Contents: Two vials containing ferritin in a proteinbased buffer with a nonmercury preservative. Prepared by spiking buffer with a defined quantity of ferritin. Refer to vial labels for the acceptable range. Volume: 0.5 ml/vial Storage: Refrigerate at 28 C Stability: 12 months in unopened vial or as indicated on label. Once opened, the controls should be used within 14 days or aliquoted and stored frozen. Avoid multiple freezing and thawing cycles. 5. WASHBUF CONC Wash Buffer Concentrate (X10) Requires Preparation Contents: One bottle containing buffer with a nonionic detergent and a nonmercury preservative. Volume: 50 ml/bottle Storage: Refrigerate at 28 C Preparation: Dilute 1:10 in distilled or deionized water before use. If the whole plate is to be used dilute 50 ml of the wash buffer concentrate in 450 ml of water. 6. ASSAYBUF Assay Buffer Ready To Use. Contents: One bottle containing a proteinbased buffer with a nonmercury preservative. Volume: 25 ml/bottle Storage: Refrigerate at 28 C V 10.0 May 15, /6
5 7. TMB SUBS TMB Substrate Ready To Use. Contents: One bottle containing tetramethylbenzidine and hydrogen peroxide in a nondmf or DMSO containing buffer. Volume: 16 ml/bottle Storage: Refrigerate at 28 C 8. TMB STOP Stopping Solution Ready To Use. Contents: One bottle containing 1 M sulfuric acid. Volume: 6 ml/bottle Storage: Refrigerate at 28 C ASSAY PROCEDURE Specimen Pretreatment: None. All reagents must reach room temperature before use. Calibrators, controls and specimen samples should be assayed in duplicate. Once the procedure has been started, all steps should be completed without interruption. 1. Prepare working solutions of the antiferritinhrp conjugate and wash buffer. 2. Remove the required number of well strips. Reseal the bag and return any unused strips to the refrigerator. 3. Pipette 20 μl of each calibrator, control and specimen sample into correspondingly labelled wells in duplicate. 4. Pipette 200 μl of the conjugate working solution into each well (We recommend using a multichannel pipette). 5. Incubate on an orbital shaker (approx. 600 rpm) or linear shaker (approx. 200 rpm) for 30 minutes at room temperature 6. Wash the wells 5 times with 300 μl of diluted wash buffer per well and tap the plate firmly against absorbent paper to ensure that it is dry. (The use of a washer is recommended). 7. Pipette 150 μl of TMB substrate into each well at timed intervals. 8. Incubate on an orbital shaker (approx. 600 rpm) or linear shaker (approx. 200 rpm) for 1015 minutes at room temperature (or until calibrator F attains dark blue colour for desired OD). 9. Pipette 50 μl of stopping solution into each well at the same timed intervals as in step Read the plate on a microwell plate reader at 450 nm within 20 minutes after addition of the stopping solution. * If the OD exceeds the upper limit of detection or if a 450nm filter is unavailable, a 405 or 415 nm filter may be substituted. The optical densities will be lower, however, this will not affect the results of patient/control samples. CALCULATIONS 1. Calculate the mean optical density of each calibrator duplicate. 2. Calculate the mean optical density of each unknown duplicate. 3. Subtract the mean absorbance value of the 0 calibrator from the mean absorbance values of the calibrators, control and serum samples. 4. Draw a calibrator curve on loglog paper with the mean optical densities on the Yaxis and the calibrator concentrations on the Xaxis. If immunoassay software is being used, a 4parameter or 5parameter curve is recommended. 5. Read the values of the unknowns directly off the calibrator curve. 6. If a sample reads more than 800 ng/ml then dilute it with calibrator A at a dilution for more than 1:8. The result obtained should be multiplied by the dilution factor. V 10.0 May 15, /6
6 TYPICAL TABULATED DATA Sample data only. Do not use to calculate results. TYPICAL CALIBRATOR CURVE Sample curve only. Do not use to calculate results. Calibrator Mean OD Value (ng/ml) A B C D E F Unknown PERFORMANCE CHARACTERISTICS SENSITIVITY The limit of detection (LoD) was determined from the analysis of 40 samples of the blank and a low value sample. LoD = 0.44 ng/ml. INTRAASSAY PRECISION Three samples were assayed ten times each on the same calibrator curve. The results (in ng/ml) are tabulated below: Sample Mean SD CV% INTERASSAY PRECISION Three samples were assayed ten times over a period of four weeks. The results (in ng/ml) are tabulated below: Sample Mean SD CV% RECOVERY Spiked samples were prepared by adding defined amounts of ferritin to three patient serum samples. The results (in ng/ml) are tabulated below: Sample Obs.Result Exp.Result Recovery% 1 Unspiked Unspiked Unspiked V 10.0 May 15, /6
7 LINEARITY Three patient serum samples were diluted with calibrator A. The results (in ng/ml) are tabulated below: Sample Obs.Result Exp.Result Recovery% 1 1:2 1:4 1:8 1:16 2 1:2 1:4 1:8 1:16 3 1:2 1:4 1:8 1: COMPARATIVE STUDIES The Ferritin ELISA kit (x) was compared with a competitors coated tube RIA kit (y). The comparison of 29 serum samples yielded the following linear regression results: y = 1.03 x R 2 = 0.97 EXPECTED NORMAL VALUES As for all clinical assays each laboratory should collect data and establish their own range of expected normal values. Group Range (ng/ml) Healthy normal males and females REFERENCES 1. Ng RH, et al. Three Commercial Methods for Serum Ferritin Compared and the HighDose Hook Effect 2. Eliminated. Clin Chem. 1983; 29(6): Vernon AP, et al. Ferritin: A Reliable Assay for Detection of Iron Defi ciency. The Ligand Quarterly. 1980; 3(4): Goldie DJ, Thomas MJ. Measurement of Serum Ferritin by Radioimmunoassay. Ann Clin Biochem. 1978; 15(2): Drysdale JW. Ferritin as a Tumor Marker. J Clin Immunoassay. 1983; 6(3): Luxton AW, et al. A Radioimmunoassay for Serum Ferritin. Clin Chem. 1977; 23(4): Bolton AE, et al. Three Different Radioiodination Methods for Human Spleen Ferritin Compared. Clin Chem. 1979; 25(10): Gonyea LM, et al. Comparison of Three Procedures for Isolating Human Ferritin, for Use as a Standard in Immunoradiometric Assay. Clin Chem. 1976; 22(4): Sukanya L, et al. Sensitive Sandwich Enzyme Immunoassay for Serum Ferritin on Microtitre Plates. Ann Clin Biochem. 1981; 18(Pt 1): V 10.0 May 15, /6
8 Symbols / Symbole / Symbôles / Símbolos / Símbolos / Σύμβολα REF LOT CONC LYO IVD Cat.No.: / Kat.Nr.: / No. Cat.: / Cat.No.: / N.º Cat.: / N. Cat.: / ΑριθμόςΚατ.: LotNo.: / ChargenBez.: / No. Lot: / LotNo.: / Lote N.º: / Lotto n.: / Αριθμός Παραγωγή: Use by: / Verwendbar bis: / Utiliser à: / Usado por: / Usar até: / Da utilizzare entro: / Χρησιμοποιείται από: No. of Tests: / Kitgröße: / Nb. de Tests: / No. de Determ.: / N.º de Testes: / Quantità dei tests: / Αριθμός εξετάσεων: Concentrate / Konzentrat / Concentré / Concentrar / Concentrado / Concentrato / Συμπύκνωμα Lyophilized / Lyophilisat / Lyophilisé / Liofilizado / Liofilizado / Liofilizzato / Λυοφιλιασμένο In Vitro Diagnostic Medical Device. / InvitroDiagnostikum. / Appareil Médical pour Diagnostics In Vitro. / Dispositivo Médico para Diagnóstico In Vitro. / Equipamento Médico de Diagnóstico In Vitro. / Dispositivo Medico Diagnostico In vitro. / Ιατρική συσκευή για InVitro ιάγνωση. Evaluation kit. / Nur für Leistungsbewertungszwecke. / Kit pour évaluation. / Juego de Reactivos para Evaluació. / Kit de avaliação. / Kit di evaluazione. / Κιτ Αξιολόγησης. Read instructions before use. / Arbeitsanleitung lesen. / Lire la fiche technique avant emploi. / Lea las instrucciones antes de usar. / Ler as instruções antes de usar. / Leggere le istruzioni prima dell uso. / ιαβάστε τις οδηγίες πριν την χρήση. Keep away from heat or direct sun light. / Vor Hitze und direkter Sonneneinstrahlung schützen. / Garder à l abri de la chaleur et de toute exposition lumineuse. / Manténgase alejado del calor o la luz solar directa. / Manter longe do calor ou luz solar directa. / Non esporre ai raggi solari. / Να φυλάσσεται μακριά από θερμότητα και άμεση επαφή με το φως του ηλίου. Store at: / Lagern bei: / Stocker à: / Almacene a: / Armazenar a: / Conservare a: / Αποθήκευση στους: Manufacturer: / Hersteller: / Fabricant: / Productor: / Fabricante: / Fabbricante: / Παραγωγός: Caution! / Vorsicht! / Attention! / Precaución! / Cuidado! / Attenzione! / Προσοχή! Symbols of the kit components see MATERIALS SUPPLIED. Die Symbole der Komponenten sind im Kapitel KOMPONENTEN DES KITS beschrieben. Voir MATERIEL FOURNI pour les symbôles des composants du kit. Símbolos de los componentes del juego de reactivos, vea MATERIALES SUMINISTRADOS. Para símbolos dos componentes do kit ver MATERIAIS FORNECIDOS. Per i simboli dei componenti del kit si veda COMPONENTI DEL KIT. Για τα σύμβολα των συστατικών του κιτ συμβουλευτείτε το ΠΑΡΕΧΟΜΕΝΑ ΥΛΙΚΑ. COMPLAINTS: Complaints may be submitted initially written or vocal. Subsequently they need to be filed including the test performance and results in writing in case of analytical reasons. WARRANTY: The product is warranted to be free from material defects within the specific shelf life and to comply with product specifications delivered with the product. The product must be used according to the Intended use, all instructions given in the instructions for use and within the product specific shelf life. Any modification of the test procedure or exchange or mixing of components of different lots could negatively affect the results. These cases invalidate any claim for replacement. LIMITATION OF LIABILITY: IN ALL CIRCUMSTANCES THE EXTENT OF MANUFACTURER S LIABILITY IS LIMITED TO THE PURCHASE PRICE OF THE KIT(S) IN QUESTION. IN NO EVENT SHALL MANUFACTURER BE LIABLE FOR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING DAMAGES FOR LOST PROFITS, LOST SALES, INJURY TO PERSON OR PROPERTY OR ANY OTHER INCIDENTAL OR CONSEQUENTIAL LOSS. IBL International GmbH Flughafenstr. 52A, Hamburg, Germany Tel.: + 49 (0) Fax: 11 IBL@IBLInternational.com WEB: Symbols Version 4.5 /